Abstract of the parental R01 Here we propose to identify and characterize a new class of signaling molecules critical for a core mechanism of cellular communication, i.e., signaling via trimeric G proteins. Trimeric G proteins play a pivotal role in a signal transduction mechanism that is conserved from yeast to humans and that constitutes the target for >25% of marketed drugs. Despite major advances in the last decades, the understanding of this signaling mechanism remains incomplete? the network of regulators that control trimeric G proteins has expanded beyond what the traditional view of this signaling pathway proposes. This has made evident that the regulation of trimeric G proteins is more complex than previously appreciated and that novel therapeutic targets may arise from the discovery of alternative mechanisms of G protein regulation. Our goal is to further the understanding of the G protein regulatory network by identifying a new family of activators, dissecting the molecular basis of their coupling to G proteins and characterizing the basic molecular mechanisms by which the prototype member of this family promotes cancer progression towards metastasis. Based on previous observations by us and others we hypothesize that a new family of non-receptor G protein activators is defined by the presence of a guanine nucleotide exchange factor (GEF) motif and that these activators assemble alternative G protein signaling circuits involved in disease. These new activators are not necessarily membrane proteins and may work in lieu of or in parallel to the canonical activators, i.e., G protein-coupled receptors (GPCRs), thereby representing a detour from the classical view of this signaling pathway. The experiments in SA#1 are designed to identify and characterize novel non-receptor G protein activators with a GEF motif by using bioinformatics, high-throughput peptide array screening and functional assays in yeast. We have validated this overall approach by identifying and partially characterizing the physiological function for some of the predicted candidates. In SA#2 we propose experiments to understand the structural basis for how this new family of non-receptor GEFs bind and activate G proteins. Taken together, these studies will determine the basic molecular principles that define a whole new family of G protein regulators and will also generate the tools required for future mechanistic studies on them. Experiments proposed in SA#3 test the general hypothesis that these non-receptor GEFs assemble alternative signaling circuits in disease for the particular case of GIV, the prototype member of this new family of GEFs. Our work to date suggests a model in which GIV-dependent activation of G proteins is required to assemble a signaling pathway linking stimulation of integrins by extracellular matrix proteins to the acquisition of pro-metastatic features by tumor cells. In summary, we hope to unravel a new general mechanism of G protein regulation with broad implications in disease by combining discovery-based and targeted mechanistic studies.